The cytostatin I of the present invention has been putatively identified as a growth inhibitory protein. This identification has been made as a result of amino acid sequence homology to mammary-derived growth inhibitor (MDGI) and direct measurements on cell growth.
Mammary-derived growth inhibitor (MDGI) is a cell growth inhibitor and differentiation factor firstly purified mammary carcinoma cells Ehrlich ascites, and then from cows milk and bovine mammary gland (Grosse et al. 2 references). MDGI inhibits proliferation of mammary epithelial cell lines in a dose-dependent and reversible manner. Maximal inhibition of cell proliferation by purified MDGI is in the range of 35 to 50%. In these cells half-maximal inhibition was obtained with about 10.sup.-10 M MDGI (1 ng/ml). Inhibition was abolished by simultaneously adding epidermal growth factor (EGF), insulin. MDGI also inhibits the proliferation of several other permanent mammary carcinoma cell lines. MDGI has been shown to be immunologically related to a fibroblast growth inhibitor.
Peptides that locally signal growth cessation and stimulate differentiation of the developing epithelium are very important for mammary gland development. Recombinant and wild-type forms of mammary-derived growth inhibitor (MDGI) and heart-fatty acid binding protein (FABP), which belong to the FABP family, specifically inhibit growth of normal mouse mammary epithelial cells (MEC) and promote morphological differentiation, stimulates its own expression and promotes milk protein synthesis. Selective inhibition of endogenous MDGI expression in MEC by antisense phosphorothioate oligonucleotides suppresses appearance of alveolar end buds and lowers the beta-casein level in organ cultures. Furthermore, MDGI suppresses the mitogenic effects of EGF, and EGF antagonizes the activities of MDGI. Finally, the regulatory properties of MDGI can be fully mimicked by an 11-amino acid sequence, represented in the COOH terminus of MDGI and a subfamily of structurally related FABPs. MDGI is the first known growth inhibitor which promotes mammary gland differentiation. The amount of MDGI increased dramatically with the onset of lactation after delivery. Recent studies shows that a new posttranslational processing form of MDGI, MDGI 2, not present in lactation, was found in the bovine gland during pregnancy. (Brandt et al, Biochem Biophy Res Comm Vol 189, p 406, Nov. 30, 1992) To date, bovine, rat and mouse MDGI have been identified but no human MDGI or MDGI-like protein.
There is no sequence homology between MDGI and other known growth inhibitors. Thus, along with interferons, transforming growth factors .beta., and tumor necrosis factors, MDGI is one of the few naturally occurring growth inhibitors for mammary epithelium identified so far. Sequence analysis revealed extensive sequence homology of MDGI to a family of low molecular mass hydrophobic ligand-binding proteins, among them a fatty acid-binding protein (FABP) from brain and heart, myelin P2, a differentiation associated protein in adipocytes (p422), gastrotropin, and the cellular retinoic acid-binding protein (CRABP). These proteins basically share two properties in common: they bind hydrophobic ligands such as long-chain fatty acids, retinoids, and eicosanoids, and they are expressed in a differentiation-dependent manner in mammary gland, heart, liver, brain, or intestine. All these proteins act intracellularly except MDGI and gastrotropin, which act extracellularly in vitro. The C-terminus of MDGI residues 126-130 are identical to residues 108-112 of bovine growth hormone. This stretch of amino acids is part of a sequence of growth hormone that is essential for its biological activity. Synthetic peptides corresponding to the MDGI-sequence, residue 121-131 mimic the effects of MDGI. The functions of these MDGI proteins are not yet well-defined, although a role in fatty acid transport, sequestration, or metabolism has been widely discussed. Interaction with as yet unknown hydrophobic ligands might play a functional role in the mechanism of growth inhibition excerted by MDGI. It is proposed that MDGI may act in an autocrine manner as a growth inhibitor, however, MDGI lack a signal sequence for membrane translocation, most of MDGI has an intracellular localization. With regard to the secretion, an analogy might exit to other growth factors that also lack a signal sequence like FGF and PG-ECGF. In those cases cell damage as a possible way of secretion, or the existence of related factors with a signal sequence as a physiological ligands of the respective surface receptors, have been discussed.
Among other activities, MDGI reportedly may inhibit c-fos, c-myc and c-ras expression . MDGI has differentiation-promoting activity on mouse pluripotent embryonic stem cells and supports the commitment of undifferentiated ESC for neural differentiation. It is also suggested that MDGI may be involved in the regulation of endothelial cell proliferation.
MDGI inhibits the induction of supersensitivity of neonatal rat heart muscle cells for beta-adrenergic receptors by lipoxygenase metabolites and various agents. The inhibitory activity of MDGI related to the induction of supersensitivity for hydrophilic beta-adrenergic agonists might point to a physiological role for a close relative of MDGI--the cardiac fatty acid-binding protein (H-FABP). One function of H-FABP could be to protect, the heart, under pathophysiological conditions, from lipoxygenase metabolites causing supersensitivity of beta-adrenergic receptors. Thus, H-FABP may be a physiological modulator of beta-adrenergic responses in the cardiac muscle.
In accordance with one aspect of the present invention, there is provided a novel mature polypeptide which is Cytostatin I, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptide of the present invention is of human origin.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding human cytostatin I, including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptide by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a human cytostatin I nucleic acid sequence, under conditions promoting expression of said protein and subsequent recovery of said protein.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes, for example, as a cell growth inhibitor and as to cause differentiation stimulatory activity on various responsive types of tissues and cells.
In accordance with yet a further aspect of the present invention, there is also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to human cytostatin I sequences.
In accordance with yet a further aspect of the present invention, there are provided antibodies against such cytostatin I polypeptides.
In accordance with another aspect of the present invention, there are provided cytostatin I agonists which mimic Cytostatin I and bind to the cytostatin I receptors to elicit growth inhibitory responses or which stimulate differentiation-promoting activity on progenitory cell types.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, in the treatment of excessive inhibition of cell or tissue growth or inappropriate differentiation stimulatory activity.
There is a need for a human MDGI-like protein and the gene encoding it. These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.